Overview

  • Product name

    Human HB EGF ELISA Kit
  • Detection method

    Colorimetric
  • Sample type

    Cell culture supernatant, Plasma
  • Assay type

    Sandwich (quantitative)
  • Sensitivity

    < 20 pg/ml
  • Range

    16.38 pg/ml - 4000 pg/ml
  • Recovery

    90 %

    Sample specific recovery
    Sample type Average % Range
    Cell culture supernatant 81.22 73% - 90%
    Plasma 93.73 84% - 103%

  • Assay duration

    Multiple steps standard assay
  • Species reactivity

    Reacts with: Human
  • Product overview

    Abcam’s HB EGF Human ELISA (Enzyme-Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of Human HB EGF in plasma and cell culture supernatants. (Human HB EGF concentration is pretty low in normal plasma, it may not be detected in this assay).


    This assay employs an antibody specific for Human HB EGF coated on a 96-well plate. Standards and samples are pipetted into the wells and HB EGF present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-Human HB EGF antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of HB EGF bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.


    We have not been able to detect the endogenous Human HB-EGF in normal serum with ab100531, only in serum spiked with Human HB-EGF.

  • Notes

    Optimization may be required with urine samples

  • Tested applications

    Suitable for: Sandwich ELISAmore details
  • Platform

    Microplate

Properties

Applications

Our Abpromise guarantee covers the use of ab100531 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Sandwich ELISA Use at an assay dependent concentration.

Images

  • Representative standard curve using ab100531

  • Representative standard curve using ab100531

Protocols

References

This product has been referenced in:

  • Wang X  et al. Sphingosine 1-Phosphate Activation of EGFR As a Novel Target for Meningitic Escherichia coli Penetration of the Blood-Brain Barrier. PLoS Pathog 12:e1005926 (2016). Read more (PubMed: 27711202) »
  • Hutchinson KE  et al. ERBB activation modulates sensitivity to MEK1/2 inhibition in a subset of driver-negative melanoma. Oncotarget 6:22348-60 (2015). Read more (PubMed: 26084293) »
See all 2 Publications for this product

Customer reviews and Q&As

1-10 of 10 Abreviews or Q&A

Answer



1. Increase amount of biotinylated ab (by 1.5 fold or so – too much may increase background)
2. Incubate biotinylated ab overnight at 4 degrees C
3. Increase amount of HRP-strep (by about 1.5 fold or so – too much may increase background)
4. Concentrate sample (using a spin column)
Please note: it’s best to try just one of these modifications at a time. Implementing too many of these changes at once may cause high background.
If the customer continues to get low absorbance values for their samples, the unfortunate assessment may be the levels of HB-EGF are simply too low for solid detection (below or close to the sensitivity of the ELISA).

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Answer

Thank you for your interest in our ELISA kits. Each kit comes with a microplate that is already coated with an antibody specific for the the protein of interest, so you would not be able to use one plate to detect all three proteins (EGF, TGF, betacellulin). The most cost effective approach for you might be an indirect ELISA, which requires coating the sample onto the plate, then detecting with an antibody specific for the protein of interest and then detecting that with an anti-IgG secondary antibody conjugated to an enzyme such as HRP. Please let me know if you have any questions.

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Answer

Thank you for contacting us.

I am very sorry to hear these kits failed to give the expected results. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

In order to better understand the problem, I’d like to gather further information. I would really appreciate if you could please clarify the following:

- What are the lot numbers for both kits and when where they received? (I would very much appreciate if you could please provide the order number as well).

- How were the kits stored and for how long? And how were the standards stored after reconstitution?

- Did you follow the standard preparation guidelines mentioned in the kit protocols?

I am happy to investigate this case for you, and the event that the kits sent happened to be faulty, I will replace o reimburse it as stated by the Abpromise guarantee.

I look forward to receiving your reply. Have a nice day.

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Answer

Thank you for the updates.

We rarely recommend running plasma or serum samples undiluted, as matrix effects are more likely to occur. For this kit (ab100531: HB EGF Human ELISA Kit), we recommend a 2-fold dilution of plasma or serum. This may help to dilute out the interfering components of the sample.

The problem with trying to reduce the background and also increase the sample signal strength is that these two factors counteract each other. For instance, extra washing will lower the background but will also decrease the overall plate signal, including the samples. On the other hand, performing the option overnight standard/sample incubation will increase the standard/sample signals but will likely also increase the background to some extent.

As you know, the sensitivity of this kit is 20 pg/ml which is actually very sensitive. However, for the customer to expect the kit to accurately and consistently detect samples which are near the sensitivity limit I think is not a practical expectation. As with all ELISA kits, sample ODs need to fall within the middle of the standard curve for the most accurate and consistent results. Sample ODs which fall on the extreme ends of the curve can be skewed by background noise or saturation and these resulting measurements would have to be viewed skeptically. This is true for all ELISA kits and for the most part, immunoassays in general.

I would like to emphasize though that this is strictly a sample dependent issue (the HB-EGF levels in their samples are likely just very low) and the kit looks to have worked as we would expect.

Although the background is not too high, I do believe the it can be lowered. Attached are some tips. For cases like these, it is important that the customer makes sure that their samples are centrifuged very well before loading onto the assay to help remove any particles or particulates that could be inhibiting detection. Making sure that the wash buffer is completely removed from all of the wells after each step is also important to keep the background low and increase the chances of getting the maximum sensitivity.

If all else fails, the customer may want to consider concentrating their sample in a non-denaturing method (for example, using a chilled spin column) or using a different sample type such as plasma or another body fluid. Ultimately they may have to report that the HB-EGF concentration in these samples is below the detection limit, which again is pretty low at 20 pg/ml.

I hope this will be useful for your customer.

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Answer

Muchas gracias por contactarnos. Le pido disculpas por la espera.

El kit al que se refiere es valido para detectar HB EGF humano en suero, plasma y sobrenadante de cultivo celular. En el protocolo adjunto en la página web se indican los pasos a seguir para realizar el ELISA, de todas formas, en nuestra página web encontrará el protocolo estándar para realizar un ensayo ELISA:

https://www.abcam.com/index.html?pageconfig=popular_protocols

Respecto a las referencias bibliográficas solicitadas me temo que toda la información disponible se encuentra en las datasheet de los productos en nuestra página web.

Es política de Abcam que toda la información disponible sobre nuestros productos es compartida y accesible a sus usuarios, mas aun tratándose del caso de publicaciones. Siento que para este kit no haya más referencias.

En cualquier caso todos nuestros productos tienen una garantía (Abpromise) de 6 meses, y en caso de no funcionar como se especifica en su datasheet, se os reemplazará el producto de forma gratuita o se reembolsará el importe del mismo.

Respecto a tiempos de entrega y al envío del protocolo mi compañera de Servicio al Cliente ya le facilitó la información. De todas formas, si requiriese más información, un presupuesto, o tuviera cualquier otra consulta, por favor, no dude en volver a contactarnos.

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Answer

Thank you for the enquiry regarding ab100531 and for sending the customer’s data/comments.

Please find enclosed the attached file for the recent analysis. As you can see, the standard curves obtained have good linearity and very low CV%. The background (˜0.29) is a little higher than whatwe would expect but it is still not too much higher than what we normally have.

It appears that some of the samples were very close to or even below the average blank OD. Please note that HB-EGF levels in normal human serum are very low. In fact, we have tested the normal human serum levels of HB-EGF and found to be undetectable.

However, this does not necessarily exclude samples that are below the sensitivity of the kit (20 pg/ml) but it is important to realize that this kit is more applicable for diseased (pathological) or treated samples.

Lowering the background may help detect the levels of HB-EGF in more of the samples. Here are a few tips for the customer to keep in mind, which may help to minimize background issues:

Too much HRP-Streptavidin may cause background. Briefly centrifuge the vial of HRP-streptavidin concentrate (Item G) and pipette up and down to mix gently before using, since precipitation may form during storage.
Too much detection antibody may also contribute to high background. Make sure of correct dilution fold of both the biotinylated antibody (Item F) and HRP-Streptavidin. Both should be diluted with 1x Assay Diluent B.
Long incubation times in some steps (i.e. overnight incubation) can cause high background.
And finally, the wash steps may introduce background: if the plate is insufficiently washed, or if the wash buffer itself was contaminated. Wash buffer must be removed completely from the wells after each wash. We recommend using an automated plate washer or multi-channel pipette. Squirt bottles are not advised.



Though you have kindly provided some details, it would be much appreciated if I could get some more information which would help me identify the source of the problem.

A) What is the sample type?

B) How were the samples prepared? Diluted?

C) Were any overnight incubations performed?

Please let me know if you need further assistance.

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Answer

Thank you for your enquiry regarding ab100531. I am very sorry to hear that your customer having problems with this kit.

The batch number you have provided (81802) does not match to our record. Could you please check it again and confirm the correct batch number. It should start with GR and a number with a hyphen.

Would you be so kind to provide the readings (values) for the blank and for the samples as well? Preferably, could you please present the data in Excel format?

I look forward to hearing from you soon.

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Answer

Thank you for your call yesterday and for your questions. I have been in touch with my colleague in the lab, and have received more information about ab100531.

The standard provided in ab100531 is expressed in baculovirus, and it corresonds to the N-terminal 148 amino acids. I have confirmed that the capture antibody is monoclonal, the detection antibody is polyclonal, and both were raised against the standard.

Unfortunately we do not have any recovery data available for urine samples, and this sample type has not been thoroughly tested by the lab.

I hope this information will be useful, but if you have any further questions please let me know and I'll be happy to help.

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Answer

Thank you for contacting Abcam.

Please find the answers to your questions below:

1. are the coating antibody and the detection antibody a polyclonal or
monoclonal antibodies?Capture antibody = monoclonal
Detection antibody = polyclonal

2. are the coating antibody and the detection antibody raised against
recombinant protein or a purified protein? And what was it raised in.. CHO
or insect cells?
The immunogen for both antibodies was a Sf21(insect cell line) recombinant human HB-EGF protein
Please let me know if there is anything else I can help you with.

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